Effective hypothermic storage of human pluripotent stem cell-derived cardiomyocytes compatible with global distribution of cells for clinical applications and toxicology testing

The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (\u3e70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs

Morpho-Rheological Fingerprinting of Rod Photoreceptors Using Real-Time Deformability Cytometry

Distinct cell-types within the retina are mainly specified by morphological and molecular parameters, however, physical properties are increasingly recognized as a valuable tool to characterize and distinguish cells in diverse tissues. High-throughput analysis of morpho-rheological features has recently been introduced using real-time deformability cytometry (RT-DC) providing new insights into the properties of different cell-types. Rod photoreceptors represent the main light sensing cells in the mouse retina that during development forms apically the densely packed outer nuclear layer. Currently, enrichment and isolation of photoreceptors from retinal primary tissue or pluripotent stem cell-derived organoids for analysis, molecular profiling, or transplantation is achieved using flow cytometry or magnetic activated cell sorting approaches. However, such purification methods require genetic modification or identification of cell surface binding antibody panels. Using primary retina and embryonic stem cell-derived retinal organoids, we characterized the inherent morpho-mechanical properties of mouse rod photoreceptors during development based on RT-DC. We demonstrate that rods become smaller and more compliant throughout development and that these features are suitable to distinguish rods within heterogenous retinal tissues. Hence, physical properties should be considered as additional factors that might affect photoreceptor differentiation and retinal development besides representing potential parameters for label-free sorting of photoreceptors

The effects of orange Citrus reticulata extracts towards vertigoand dizziness of EEGAFI Students

Dizziness and imbalance are common conditions affecting people of all ages in different walks of life, particularly the students. This study was performed to describe and determine the potential effect of orange peel on dizziness or vertigo towards the students in EEGAFI. The researchers will require a field to conduct a sort of semi- experiment but not performed in clinical sphere of producing the finished extract of Citrus reticulata. The researchers have chosen the students of ECT Excellencia Global Academy Foundation, Inc. (EEGAFI) in a targeted sample of the population. Since the location where this survey was held provided “hybrid blended learning,” the research team also used notes and questionnaires via Google Forms, and some respondents found it convenient to respond in their own homes, making it simple for them to assume responsibility as respondents in this research study. In total, 11 students from various strands of the EEGAFI in grades 11 and 12 who had previously experienced vertigo or dizziness participated in this study. There were 11 STEM a (bravery), 11 STEM B (perseverance), and 12 UNITY (Graph 1). The result of the study found out that Citrus reticulata had a statistically significant effect on the study participants’ dizziness and vertigo experiences. It is imperative to maintain the physical fitness of EEGAFI students. The results suggest that dizziness and vertigo have a real impact on a student’s ability to develop and improve their activities and academic performance. Students who experience the pain and distractions of dizziness will likely experience negative emotions, burdens, and tension that cause terrible results in class. Due to their inability to properly comprehend their ideas and original works, they will probably also get a lesser grade

Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia

Modeling of surface area and pore volume of activated carbons prepared from renewable and low cost precursors

In this study, methylene blue and iodine numbers were used to optimize the conditions of preparation of activated carbons from renewable and low cost precursors. Standard methods of determining iodine number and methylene blue absorption were applied. These were used to model surface area and pore volume. Each carbon prepared under different experimental conditions was characterized with the goal of using the data as a response factor in the determination of optimum preparation conditions for each carbon

Multimodal profiling of the transcriptional regulatory landscape of the developing mouse cortex identifies Neurog2 as a key epigenome remodeler.

How multiple epigenetic layers and transcription factors (TFs) interact to facilitate brain development is largely unknown. Here, to systematically map the regulatory landscape of neural differentiation in the mouse neocortex, we profiled gene expression and chromatin accessibility in single cells and integrated these data with measurements of enhancer activity, DNA methylation and three-dimensional genome architecture in purified cell populations. This allowed us to identify thousands of new enhancers, their predicted target genes and the temporal relationships between enhancer activation, epigenome remodeling and gene expression. We characterize specific neuronal transcription factors associated with extensive and frequently coordinated changes across multiple epigenetic modalities. In addition, we functionally demonstrate a new role for Neurog2 in directly mediating enhancer activity, DNA demethylation, increasing chromatin accessibility and facilitating chromatin looping in vivo. Our work provides a global view of the gene regulatory logic of lineage specification in the cerebral cortex

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

To fully explore the potential of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4 degrees C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness

Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium

<div><p>A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.</p> </div

Characterization of the hESC-derived RPE sheets.

<p>(A) Immunostaining of ZO-1 (red) on hESC-derived RPE cells at Day 30 indicated the presence of tight junctions. Pigmented cells displayed polygonal shape. (B–D) Electron microscopic analyses of hESC-derived RPE cells at Day 50. hESC-derived RPE cells had abundant apical microvilli (mv), melanin granules (mel) in the apical half and the nucleus (nu) in their basal half. A basement membrane (bm) was visible. Tight junctions (tj) and desmosomes (de) could be found. (E) The transepithelial resistance (TER) of hESC-derived RPE progenitor or RPE cells increases during differentiation. (F) Cross-sections through the pigmented cell sheet immunostained for RPE65 and BESTROPHIN. hESC-derived RPE cells at Day 40 expressed the mature RPE cell markers RPE65 and BESTROPHIN. (G) hESC-derived RPE cells exited the cell cycle by Day 30 as demonstrated by RPE cultures pulse labeled with EdU at Day 7, 15 and 30. EdU incorporation by proliferating cells was observed at Day 7 and 15, but rarely observed at Day 30.</p

Transplantation of hESC-derived RPE cells effectively rescues photoreceptors in RCS rats.

<p>(A, B) Sections of transplanted retina in the central region adjacent to the injection site shown in DIC image (A) and immunostained to identify human nuclei (red) and DAPI (blue) (B). Donor RPE cells (red) were localized in the sub-retinal space integrated into the host RPE monolayer and generated monolayer-like structures (A, B, arrows). The ONL overlying the donor RPE monolayer was well preserved and contained 5–6 rows of nuclei (B, arrow heads). (C) Electron microscopic analyses of transplanted RPE cells. The dotted line outlined one RPE cell. Typical polarized RPE morphology with foldings at the basal membrane, a nucleus located at the basal side (labeled by N) and apically located melanin-containing melanosomes (some labeled by M) were observed. (D, E) Representative fluorescence images of central or peripheral region in the hESC-derived RPE cell transplanted retina. Dashed box showed the ONL. Following transplantation of donor RPE cells, central regions of the retina showed an increased ONL thickness with an average of 5–6 rows of nuclei (D) in contrast to the periphery with 1–2 rows of nuclei (E). (F) Quantitative data for the effect of different transplanted cells on ONL thickness. (G) Quantitative data for the protected area of ONL in the human RPE and fibroblast cell transplanted retina. In panels (F) and (G), 6 RCS rats transplanted with human ESC-derived RPE cells, 5 with human fibroblasts, 5 sham-injected, and 6 untreated controls were quantified. Data were presented as means±SD. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. n.s.: not significant, *: P<0.05, **: P<0.01, ***: P<0.001. Nuclei were counterstained with DAPI. Scale bars, 10 µm (A, B), 5 µm (C), 20 µm (D, E).</p